Thiol carboxylic acid derivatives and their use as colagenase inhibitors

ABSTRACT

Novel compounds of the formula (I), processes for their preparation and their use as collagenase inhibitors are described: ##STR1## in which R 1  is hydroxy; alkoxy; aryloxy; aralkyloxy; --NR 6  R 7  where R 6  and R 7  are hydrogen or alkyl, or R 6  and R 7  together with the N-atom to which they are bonded form a 5- to 7-membered ring with an optional heteroatom; --NHCH(R 8 )COR 9  where R 8  is hydrogen; alkyl optionally substituted by hydroxy, alkoxy, --NR 6  R 7 , quanidine, CO 2  H, CONH 2 , SH, or S-alkyl; or CH 2  -AR where Ar is optionally substituted aryl; and R 9  is hydroxy, alkoxy or --NR 6  R 7 . 
     R 2  is hydrogen or acyl. 
     R 3  is C 3-6  alkyl. 
     R 4  is hydrogen; alkyl; --CH 2  R 10  where R 10  is optionally substituted phenyl or heteroaryl; or --CH(R 12 )O--R 11  where R 11  is hydrogen; alkyl; or --CH 2  Ph where Ph is optionally substituted phenyl; and R 12  is hydrogen or alkyl. 
     R 5  is hydrogen; alkyl; or --CH(R 13 )COR 14  where R 13  is hydrogen or alkyl; and R 14  is hydroxy, alkoxy or --NR 6  R 7 .

The present invention relates to novel thiol-carboxylic acidderivatives, processes for their preparation and their use in medicine.In particular, the present invention relates to their use as collagenaseinhibitors for treating arthritic and other diseases.

The range of therapeutic applications of the collagenase inhibitorsdescribed hereinafter reflects the fundamental role of collagen withinthe connective tissue matrix throughout the body, and extends to manydiseases not primarily due to collagen destruction but involving tissueremodelling, as these will also be susceptible to clinical interventionwith collagenase inhibitors. In particular, inhibition of collagenasesreleased from synovial and skin fibroblasts, chondrocytes, peripheralmononuclear cells, keratinocytes and gingival tissue, as well asinhibition of collagenase stored in polymorphonuclear leucocytes (PMNLs)should be of therapeutic value, and the present compounds are envisagedas having application against these and related mammalian collagenases.

Specifically, collagenase inhibitors will provide useful treatments forarthritic diseases such as rheumatoid arthritis and osteoarthritis, softtissue rheumatism, polychondritis and tendonitis; for bone resorptiondiseases such as osteoporosis, Paget's disease, hyperparathyroidism andcholesteatoma; for the recessive classes of dystrophic epidermolysisbullosa; for periodontal disease and related consequences of gingivalcollagenase production or of PMNL collagenase production followingcellular infiltration to inflamed gingiva; for corneal ulceration e.g.that induced by alkali or other burns, by radiation, by vitamin Edeficiency or retinoid deficiency; and for systemic chemotherapy ofcancer, where collagenase has been implicated in the neovascularizationrequired to support tumour survival and growth, and in the penetrationof tumour cells through the basement membrane of the vascular wallsduring metastasis. A collagenase inhibitor may also be of use in somepost-operative conditions such as colonic anastomosis in whichcollagenase levels are raised.

As a particular example of the therapeutic value of collagenaseinhibitors, chronic arthritic diseases lead to extensive loss of thecollagen and proteoglycan components within the cartilage and bone ofthe affected joints. Neutral metalloproteases, especially collagenasesand proteoglycanases, are currently thought to be the major enzymesinvolved.

These enzymes have been detected in extracts of synovial and cartilagetissue, and have also been extensively studied in tissue cultures ofthese organs. Apart from control of the biosynthesis or secretion of theenzymes, the most significant natural regulation of the activity ofcollagenase and proteoglycanase in the normal and diseased state, isconsidered to be the production of inhibitors such as the TissueInhibitor of Metalloproteases (TIMP) and α₂ -macroglobulin. An imbalancebetween the levels of proteolytic enzymes and natural inhibitors willallow destruction of the connective tissue components to proceed.

Restoration of the enzyme-inhibitor balance by treatment with syntheticinhibitors of collagenase thus offers a useful therapy for a wide rangeof connective tissue diseases in which collagenolytic activity is acausative or major contributory factor.

U.S. Pat. No. 4,595,700 discloses compounds of the formula (A): ##STR2##in which R₁ represents lower alkyl, phenyl or phenyl lower alkyl;

R₂ and R₄ represent lower alkyl; and

R₃ represents lower alkyl, benzyloxyalkyl, alkoxybenzyl orbenzyloxybenzyl wherein the oxyalkyl or alkoxy moiety contains 1 to 6carbon atoms and a, b and c represent chiral centres with optional R orS stereochemistry.

These compounds are described as inhibitors of collagenase, useful inthe treatment of rheumatoid arthritis and related diseases in whichcollagenolytic activity is a contributing factor.

A novel class of thiol-carboxylic acid derivatives has now beendiscovered, which are collagenase inhibitors and thus of potentialutility in the treatment of diseases in which collagenolytic activityand tissue remodelling is implicated.

According to the present invention there is provided a compound ofgeneral formula (I), or a salt, solvate or hydrate thereof: ##STR3## inwhich R₁ is --OH; alkoxy; aryloxy; aralkyloxy; --NR₆ R₇, where each ofR₆ and R₇ is hydrogen or alkyl, or R₆ and R₇ together with the nitrogenatom to which they are bonded form a 5-, 6- or 7-membered ring with anoptional oxygen, sulphur or optionally substituted nitrogen atom in thering; or a group ##STR4## where R₈ is hydrogen; alkyl optionallysubstituted by --OH, alkoxy, --NR₆ R₇, guanidine, --CO₂ H, CONH₂, SH, orS-alkyl; or --CH₂ --Ar where Ar is optionally substituted aryl; and R₉is alkoxy; OH; or --NR₆ R₇ ;

R₂ is hydrogen; or acyl, such as ##STR5## where Z is optionallysubstituted aryl; R₃ is C₃₋₆ alkyl; R₄ is hydrogen; alkyl; --CH₂ --R₁₀where R₁₀ is optionally substituted phenyl or heteroaryl; or a group##STR6## where R₁₁ is hydrogen; alkyl; or --CH₂ --Ph where Ph isoptionally substituted phenyl; and R₁₂ is hydrogen or alkyl; and

R₅ is hydrogen; alkyl; or a group ##STR7## where R₁₃ is hydrogen; oralkyl; and R₁₄ is hydroxy; alkoxy; or --NR₆ R₇.

Unless otherwise specified, each alkyl or alkoxy group is a C₁₋₈ group,more preferably C₁₋₆, and may be a straight chain or branched.

Optional substituents for aryl, phenyl and heteroaryl groups may beselected from OH, C₁₋₆ alkyl, C₁₋₆ alkoxy and halogen.

Examples of R₁ are hydroxy; C₁₋₆ alkoxy, such as methoxy, ethoxy ort-butyloxy; benzyloxy; and --NR₆ R₇ in which R₆ is hydrogen, and R₇ ishydrogen or C₁₋₈ alkyl such as methyl or ethyl; or --NR⁶ R₇ isN'-methyl-N-poperazinyl or N-morpholinyl. Other examples of R₁ are--NH--CH₂ --COOH, --NH--CH₂ --CONH₂, --NH--CH₂ --CO₂ Et, --NH--CH₂ --CO₂^(t) Bu, ##STR8##

R₁ is preferably alkoxy, such as C₁₋₄ alkoxy, especially methoxy; amino;alkylamino, especially methylamino or --NH--CH₂ --CO₂ H.

When R₂ is ##STR9## Z is preferably an optionally substituted phenylgroup.

Examples of R₂ are hydrogen; ##STR10## and benzoyl.

R₃ is preferably a C₄ alkyl group, such as n-butyl, iso-butyl orsec-butyl, especially iso-butyl.

When R₄ is --CH₂ --R₁₀ and R₁₀ is heteroaryl, values for R₁₀ include5-or 6- membered monocyclic and 9-or 10-membered bicyclic heteroaryl ofwhich 9- or 10-membered bicyclic heteroaryl is preferred.

In addition, 5-or 6-membered monocyclic and 9-or 10-membered bicyclicheteroaryl preferably contain one or two heteroatoms selected fromnitrogen, oxygen and sulphur which in the case of there being more thanone heteroatom may be the same or different. When R₁₀ is 9-or 10-membered bicyclic heteroaryl the two rings are preferably fused with one5- or 6- membered ring containing a single heteroatom.

R₄ is preferably iso-butyl; benzyl; or C₁₋₆ alkoxybenzyl, such as4-methoxybenzyl; 1-(benzyloxy)ethyl or 9- or 10- membered fused bicyclicheteroarylmethyl such as 3-indolylmethyl.

Examples of R₅ include hydrogen; alkyl, such as methyl or ethyl,preferably methyl; and 1-(methoxycarbonyl)ethyl.

The compounds of formula (I) may form salts with bases e.g. sodiumhydroxide. When a basic nitrogen atom is present, the compounds offormula (I) may form acid addition salts e.g with hydrochloric acid.Such compounds form part of the present invention.

Where compounds of formula (I), or salts thereof, form solvates such ashydrates, these also form an aspect of the invention.

The compounds of formula (I) have at least one asymmetric centre andtherefore exist in more than one stereoisomeric form. The inventionextends to all such forms and to mixtures thereof, including racemates,and diastereoisomeric mixtures.

Preferred isomers are those having the S configuration at the chiralcentre marked with an asterisk in formula (I).

The compounds of formula I or their salts, solvates or hydrates arepreferably in pharmaceutically acceptable or substantially pure form. Bypharmaceutically acceptable form is meant, inter alia, of apharmaceutically acceptable level of purity excluding normalpharmaceutical additives such as diluents and carriers, and including nomaterial considered toxic at normal dosage levels. A substantially pureform will generally contain at least 50% by weight, preferably 75%, morepreferably 90% and still more preferably 95% or 99% or more of thecompound of formula I or its salt or solvate.

One preferred pharmaceutically acceptable form is the crystalline form.

The present invention provides the compounds of formula (I) orpharmaceutically acceptable salts or solvates thereof for use as activetherapeutic agents, particularly as agents for treatment ofmusculo-skeletal disorders resulting from collagenolytic activity,particularly arthritic diseases, and tissue remodelling, and also forthe systemic chemotherapy of cancer.

The present invention also provides a process for the preparation of acompound of formula (I) in which R₂ is hydrogen, which comprisescleaving a group L from a compound of formula (II): ##STR11## wherein Lis L₁ which is a conventional sulphur protecting group or L₂ which is agroup R-S- where R is any organic residue such that the group R-S-provides a cleavable disulphide bond, and R₁, R₃, R₄ and R₅ are asdefined for formula (I).

Typically a protecting group L₁ is a substituted benzyl group, such asalkoxybenzyl e.g. 4-methoxybenzyl or an aliphatic or aryl acyl groupsuch as acetyl or benzoyl. When L₁ is acyl it is of course identical toR₂, so these compounds of formula (II) are themselves compounds of theinvention. When L is R-S- then typically the compound of formula (II) isa dimer of the compound of formula (I) in which R₂ is hydrogen.

When L is L₁, and L₁ is a substituted benzyl sulphur protecting group,such as 4-methoxy benzyl, then L₁ may be removed by treatment withmercury acetate in trifluoroacetic acid containing anisole followed byreaction with hydrogen sulphide in dimethyl formamide, in a procedureanalogous to that described in Chem. Pharm. Bull 1957, 26, 1576.

When L₁ is an acyl group it may be removed by treatment with a base, forexample aqueous ammonia or dilute aqueous sodium hydroxide, or bytreatment with an acid, for example methanolic hydrochloric acid.

When L is L₂ the dimerized compound may be split at the disulphide linkby treatment with zinc and hydrochloric acid or by passing hydrogensulphide through the solution.

Other conventional methods for removing sulphur protecting groups orcleaving disulphide bridges may also be used.

Compounds of formula (II) in which L is L₁ may be prepared by treating acompound of formula (III): ##STR12## in which R₁, R₃, R₄ and R₅ are asdefined in formula (I) with a thiol of formula (IV):

    L.sub.1 --SH                                               (IV)

in which L₁ is as defined in formula (II). When L₁ is R₂, the compoundsof formula (II) thereby produced are compounds of the invention.

Compounds of formula (II) may also be prepared by treating a compound offormula (V): ##STR13## in which L, R₁ and R₃ are as defined in formula(II), with a compound of formula (VI): ##STR14## in which R₄ and R₅ areas defined in formula (I).

The reaction is preferably carried out in the presence of a couplingagent, such as N,N-dicyclohexylcarbodiimide.

Compounds of formula (II) in which L is L₂ may also be prepared byoxidative coupling, with iodine or oxygen, of a compound of formula (I)in which R₂ is hydrogen.

Compounds of formula (II) can be converted to further compounds offormula (II) while retaining the same group L, which group in turn canbe cleaved to form compounds of the invention in which R₂ is hydrogen.

For example, those compounds of formula (II) in which R₁ is --OH may beprepared under acid conditions by hydrolysis of compounds in which R₁ isalkoxy, aryloxy or aralkyloxy or by hydrogenolysis of compounds in whichR₁ is benzyloxy or substituted benzyloxy in the presence of a catalystsuch as palladium black.

Those compounds of formula (II) in which R₁ is --NR₆ R₇ may be preparedfrom compounds in which R₁ is --OH by treating the latter compounds withan amine of formula NHR₆ R₇ in the presence of a coupling agent such asN,N-dicyclohexylcarbodiimide orN-ethyl-N'-dimethylaminopropylcarbodiimide.

Compounds of formula (II) in which R₁ is --NH--CH(R₈)--COR₉ may besimilarly prepared from compounds in which R₁ is OH by treatment withamine derivatives of formula NH₂ CH(R₈)COR₉ where R₉ is an alkoxy oramine group, followed by hydrolysis to give an R₉ hydroxy group, ifdesired.

In addition, compounds of the invention in which R₂ is acyl can beconverted to further compounds of the invention with concomitantcleavage of the acyl group to give compounds of formula (I) in which R₂is hydrogen.

For example, those compounds of formula (I) in which R₁ is OH and R₂ ishydrogen may be prepared by hydrolysis of compounds in which R₁ isalkoxy, aryloxy or aralkyloxy and R₂ is acyl under basic conditions suchas treatment with dilute sodium hydroxide.

The intermediate compounds of formula (111) may be prepared by treatinga compound of formula (VII): ##STR15## in which R₁ and R₃ are as definedin formula (I), with a compound of formula (VI) as defined above.

The thiols of formula (IV) are known compounds.

The intermediate compounds of formula (V) may be prepared by treating acompound of formula (VII), as defined above, with a thiol of formula(IV).

It may be necessary or convenient to protect the carboxyl function incompounds of formula (VII), for example by esterification, prior totreatment with the thiol of formula (IV) and subsequently remove theprotecting group under acid conditions.

The compounds of formula (VI) are either known amino acid derivatives orcan be made from these derivatives by known methods.

The intermediates of formulae (III), (V) and (VII) disclosed herein arein some forms novel compounds and form an aspect of the presentinvention as do the described processes for their preparation.

The preparation of certain compounds of formula (VII) is illustrated inthe following reaction Scheme I, using compounds in which R₃ isiso-butyl.

The starting material, compound A in Scheme I is a known compound,diethyl 3-oxopentanedioate.

The preparation of compounds of formula (V) from compounds of formula(VII) in which the carboxyl group is protected as described above isillustrated in reaction Scheme 2, using compounds in which R₁ ismethylamino, R₃ is iso butyl, L is benzoyl and the carboxyl group isprotected as a t-butyl ester. ##STR16##

Where obtainable, pharmaceutically acceptable salts of the compounds offormula (I) may be formed conventionally by reaction with theappropriate acid or base. Solvates may be formed by crystallization fromthe appropriate solvent.

As mentioned previously, the compounds of formula (I) exist in more thanone diastereoisomeric form. Where the processes of the invention producemixtures thereof, the individual isomers may be separated one fromanother by chromatography, e.g. HPLC.

Alternatively, separate diastereoisomeric compounds of formula (I) canbe obtained by using stereoisomerically pure starting materials or byseparating desired isomers of intermediates at any stage in the overallsynthetic process, and converting these intermediates to compounds offormula (I).

The present invention further provides a pharmaceutical composition,which comprises a compound of formula (I), or a pharmaceuticallyacceptable salt, solvate or hydrate thereof, and a -pharmaceuticallyacceptable carrier.

A composition of this invention is useful in the treatment of rheumatismand arthritis and in the treatment of other collagenolytic conditions.

A composition of the invention, which may be prepared by admixture, maycontain a diluent, binder, filler, disintegrant, flavouring agent,colouring agent, lubricant or preservative in conventional manner. Theseconventional excipients may be employed in conventional manner, forexample as in the preparation of compositions of related peptide enzymeinhibitors, such as the ACE inhibitor captopril.

A composition of the invention may be adapted for oral, topical,percutaneous, rectal or parenteral-intravenous, intramuscular,sub-cutaneous, intradermal or intra-articular administration, but oraladministration is preferred.

Preferably, a pharmaceutical composition of the invention is in unitdosage form and in a form adapted for use in the medical or veterinarialfields. For example, such preparations may be in a pack form accompaniedby written or printed instructions for use as an agent in the treatmentor prophylaxis of any of the disorders mentioned above.

The suitable dosage range for the compounds of the invention may varyfrom compound to compound and may depend on the condition to be treated.It will also depend, inter alia, upon the relation of potency toabsorbability and the mode of administration chosen.

The compound or composition of the invention may be formulated foradministration by any route, the preferred route depending upon thedisorder for which treatment is required, and is preferably in unitdosage form or in a form that a human patient may administer to himselfin a single dosage.

Compositions may, for example, be in the form of tablets, capsules,sachets, vials, powders, granules, lozenges, reconstitutable powders, orliquid preparations, for example solutions or suspensions, orsuppositories.

The compositions, for example those suitable for oral administration,may contain conventional excipients such as binding agents, for examplesyrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone;fillers, for example lactose, sugar, maize-starch, calcium phosphate,sorbitol or glycine; tabletting lubricants, for example magnesiumstearate: disintegrants, for example starch, polyvinylpyrrolidone,sodium starch glycollate or microcrystalline cellulose; orpharmaceutically acceptable setting agents such as sodium laurylsulphate.

Solid compositions may be obtained by conventional methods of blending,filling, tabletting or the like. Repeated blending operations may beused to distribute the active agent throughout those compositionsemploying large quantities of fillers. When the composition is in theform of a tablet, powder, or lozenge, any carrier suitable forformulating solid pharmaceutical compositions may be used, examplesbeing magnesium stearate, starch, glucose, lactose, sucrose, rice flourand chalk. Tablets may be coated according to methods well known innormal pharmaceutical practice, in particular with an enteric coating.The composition may also be in the form of an ingestible capsule, forexample of gelatin containing the compound, if desired with a carrier orother excipients. For example, in a hard gelatin capsule containing therequired amount of a compound of the invention in the form of a powderor granulate in intimate mixture with a lubricant, such as magnesiumstearate, a filler, such as microcrystalline cellulose, and adisintegrant, such as sodium starch glycollate.

Compositions for oral administration as liquids may be in the form of,for example, emulsions, syrups, or elixirs, or may be presented as a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Such liquid compositions may contain conventional additives such assuspending agents, for example sorbitol, syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminiumstearate gel, hydrogenated edible fats; emulsifying agents, for examplelecithin, sorbitan monooleate, or acacia; aqueous or non-aqueousvehicles, which include edible oils, for example almond oil,fractionated coconut oil, oily esters, for example esters of glycerine,or propylene glycol, or ethyl alcohol, glycerine, water or normalsaline; preservatives, for example methyl or propyl p-hydroxybenzoate Orsorbic acid; and if desired conventional flavouring or colouring agents.

The compounds of this invention may also be administered by a non-oralroute. In accordance with routine pharmaceutical procedure, thecompositions may be formulated, for example for rectal administration asa suppository or for parenteral administration in an injectable form.For injection, the compounds of the invention may be presented in anaqueous or non-aqueous solution, suspension or emulsion in apharmaceutically acceptable liquid, e.g. sterile pyrogen-free water or aparenterally acceptable oil or a mixture of liquids, which may containbacteriostatic agents, anti-oxidants or other preservatives, buffers orsolutes to render the solution isotonic with the blood, thickeningagents, suspending agents or other pharmaceutically acceptableadditives. Such forms will be presented in sterile unit dose form suchas ampoules or disposable injection devices or in multi- dose forms suchas a bottle from which the appropriate dose may be withdrawn or a solidform or concentrate which can be used to prepare an injectableformulation.

For topical and percutaneous administration, the preparations may alsobe presented as an ointment, cream, lotion, gel, spray, aerosol, wash orskin paint or patch.

A unit dose for inflammatory diseases will generally contain from 10 to1000 mg and preferably will contain from 10 to 500 mg, in particular 10,50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg. The compositionmay be administered once or more times a day, for example 2, 3 or 4times daily, so that the total daily dose for a 70 kg adult willnormally be in the range 10 to 3000 mg. Alternatively, in particular forinjection, the unit dose will contain from 2 to 200 mg of a compound ofthe invention and be administered in multiples, if desired, to give thedesired daily dose.

The present invention additionally provides a method of treating acollagenolytic condition such as rheumatism and/or arthritic conditions,or cancer, or other diseases in which enzyme-mediated breakdown ofconnective tissue components plays a role in mammals, such as humans,which comprises administering an effective amount of a compound offormula (I) or a pharmaceutically acceptable salt, solvate or hydratethereof, to the mammal.

The present invention also provides the use of a compound of formula (I)or a pharmaceutically acceptable salt, solvate or hydrate thereof, forthe manufacture of a medicament for use as an active therapeuticsubstance, particularly in the treatment of collagenolytic conditions,such as rheumatism, cancer, bone disorders, skin diseases, periodontaldisease or corneal ulceration, in mammals.

The following Descriptions and Examples illustrate the preparation ofcompounds of the invention and the subsequent biological dataillustrates their pharmacological activity. All temperatures areexpressed in °C.

DESCRIPTION 1 Diethyl 2-(2-methylpropyl)-3-oxopentanedioate (D1)##STR17##

Diethyl 3-oxopentanedioate (45.5 ml, 0.25 mol) was added to a suspensionof magnesium ethoxide (prepared from magnesium turnings (9 g, 0.375 mol)and iodine (0.1 g) in dry ethanol (275 ml)), and the mixture was heatedunder reflux for 90 min. 1-Bromo-2-methylpropane (54 ml, 0.5 mol) wasadded, and the reaction was heated under reflux for 16 h. Further1-bromo-2-methylpropane (27 ml, 0.25 mol) was added to the boilingmixture, and after 3 h the solvent was evaporated in vacuo. The residuewas partitioned between 2N hydrochloric acid and ether. After extractingthe aqueous phase with ether, the combined organics were washedsuccessively with water, 5% sodium bicarbonate, water and brine, thenwere dried (Na₂ SO₄) and evaporated in vacuo. The residue was distilledto give the title compound as a colourless oil (40 g, 62%), bp 118°-120°C. (1mmHg).

δ (CDCl₃): 0.9 (6H, d, J=6 Hz), 1.25 (6H, t, J=7 Hz), 1.4-1.9 (3H, m),3.5 (2H, s), 3.7 (1H, d, J=9 Hz) and 4.2 (4H, q, J=7 Hz).

DESCRIPTION 2 Diethyl 3-hydroxy-2(2-methylpropyl)pentanedioate (D2)##STR18##

Sodium borohydride (1.1 g, 29 mmol) was added to an ice-cold solution ofdiethyl 2-(2-methylpropyl)-3-oxopentanedioate (7.5 g, 29 mmol) inethanol (75 ml). The mixture was stirred at 5°-10° C. for 2 h, then 2Nhydrochloric acid (18 ml) was added slowly at 0° C., and the mixture wasstirred at room temperature for 45 min. The mixture was diluted withwater (400 ml), then was extracted with ethyl acetate (4×100 ml). Theextracts were washed with water (2 100 ml) and brine (100 ml), then weredried (Na₂ SO₄) and evaporated in vacuo to leave the title compound asan oil (7.5 g, 100%).

δ (CDCl₃): 0.9 (6H, d, J=5 Hz), 1.15 (6H, t, J=7 Hz), 1.1-1.8 (3H, m),2.2-2.7 (3H, m), 3.3 (1H, brs), 3.7-4.1 (1H, m) and 4.1 (4H, q, J=7 Hz).

DESCRIPTION 3 2- and 4-(2-Methylpropyl)pent-2-enedioic acids (D3)##STR19##

Diethyl 3-hydroxy-2-(2-methylpropyl)pentanedioate (26 g, 100 mmol) wasadded to a stirred suspension of phosphorus pentoxide (21.3 g, 150 mmol)in dry benzene (220 ml), and the mixture was heated under reflux for 3h. Water (200 ml) was added to the cooled mixture to break down thebrown gum. The layers were separated and the aqueous phase was extractedwith ether (4×150 ml). The combined organics were washed with water (150ml) and brine (300 ml), then were dried (Na₂ SO₄) and evaporated invacuo to leave a brown oil. The above oil was dissolved in a solution ofpotassium hydroxide (16.8 g, 300 mmol) in 80% ethanol (300 ml), and themixture was heated under reflux for 3 h. The ethanol was evaporated invacuo and the residue was diluted with water (400 ml), and washed withether (150 ml). The aqueous solution was acidified with 5N hydrochloricacid, then was extracted with ether (4×150 ml). The combined extractswere washed with brine (2×100 ml), then were dried (Na₂ SO₄) andevaporated in vacuo to leave a mixture of the two title compounds as anoil which slowly solidified (14.3 g, 77%). The mixture of acids was usedin the following stage without separation.

However, it was possible to separate the isomers by crystallisation fromether/pentane, which gave 2-(2-methylpropyl)pent-2-enedioic acid as asolid, m.p. 129°-135° C. (chloroform).

δ (DMSO d₆): 0.9 (6H, d, J=6 Hz), 1.3-2.0 (1H, m), 2.15 (2H, d, J=7 Hz),3.2 (2H, d, J=7 Hz), and 6.8 (1H, t, J=7 Hz).

Evaporation of the mother liquors gave 4-(2-methylpropyl)pent-2-enedioicacid as an oil.

δ (CDCl₃): 0.9 (6H, d, J=6 Hz), 1.4-2.3 (3H, m), 3.4 (1H, m), 5.85 (1H,d, J=15 Hz) and 7.0 (1H, dd, J=15, 7 Hz).

DESCRIPTION 4 2-(2-Methylpropyl)pent-2-enedicarboxylic anhydride (D4)##STR20##

A mixture of 2- and 4-(2-methylpropyl)pent-2-enedioic acids (14.3 g, 77mmol) in acetic anhydride (40 ml) was heated under reflux for 21/2 h.The acetic anhydride was evaporated in vacuo to leave a brown oil, whichwas distilled to give the title compound as a low-melting yellow solid(5.8 g, 45%), b.p. 145°-155° C. (5 mmHg).

δ (CDCl₃): 0.9 (6H, d, J=6 Hz), 1.5-2.4 (3H, m), 3.5 (2H, brd, J=4 Hz),6.5 (1H, t, J=4 Hz).

DESCRIPTION 5 4-Methoxycarbonyl-2-(2-methylpropyl)but-2-enoic acid (D5)##STR21##

2-(2-Methylpropyl)pent-2-enedicarboxylic anhydride (960 mg, 5.7 mmol)was dissolved in concentrated sulphuric acid (10 ml), then immediatelypoured into ice-cooled methanol (50 ml). The solution was diluted withwater (100 ml) and was extracted with ether (3×50 ml). The extracts werewashed with brine, then dried (MgSO₄) and the solvent was evaporated invacuo to leave the title compound as a pale yellow solid (960 mg, 84%),mp 40°-45° C. (hexane).

δ (CDCl₃): 0.9 (6H, d, J=6 Hz), 1.4-2.0 (1H, m), 2.2 (2H, d, J=6 Hz),3.55 (2H, d, J=7 Hz), 6.2 (1H, t, J=7 Hz) and 9.6 (1H, brs). ν_(max)(nujol): 1735, 1690 and 1635cm⁻¹.

DESCRIPTION 66-Methyl-4-[[[1-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-3-enoicacid, methyl ester (D6) ##STR22##

N,N-Dicyclohexylcarbodiimide (515 mg, 2.5 mmol) was added to anice-cooled solution of 4-methoxycarbonyl-2-(2-methylpropyl)but-2-enoicacid (500 mg, 2.5 mmol) and O-methyl-L-tyrosine N-methylamide (520 mg,2.5 mmol) in dry dichloromethane (30 ml), and the mixture was stirred atroom temperature for 18 h. The precipitated solid was filtered off andwashed with a little dichloromethane, then the organic solution waswashed successively with 1N hydrochloric acid (20 ml), 1N sodiumbicarbonate (20 ml), and water. The solution was dried (MgSO₄) andevaporated in vacuo. Column chromatography (30 g silica) of the residualsolid, eluting with 5% methanol/dichloromethane gave the title compound(520 mg, 53%), m.p. 142°-144° C. (dichloromethane/pentane).

δ (CDCl₃): 0.82 (3H, d, J=7 Hz), 0.83 (3H, d, J=7 Hz), 1.58 (1H, m),2.18 (2H, d, J=7 Hz), 2.72 (3H, d, J=5 Hz), 3.02 (2H, m), 3.16 (2H, d,J=7 Hz), 3.70 (3H, s), 3.78 (3H, s), 4.57 (1H, m), 5.70 (1H, brs), 6.32(1H, t, J=7 Hz), 6.45 (1H, d, J=8 Hz), 6.83 (2H, d, J=8 Hz) and 7.12(2H, d, J=8 Hz).

DESCRIPTION 7 4-Benzyloxycarbonyl-2-(2-methylpropyl)but-2-enoic acid(D7) ##STR23##

The title compound was prepared in 91% yield from benzyl alcohol by themethod Of D5.

A sample recrystallized from ether/pentane gave white needles, m.p.72-73.5. (Found: C, 69.55; H, 7.38. C₁₆ H₂₀ O₄ requires C, 69.54; H,7.30%).

δ (CDCl₃): 0.9 (6H, d, J=6 Hz), 1.4-2.0 (1H, m), 2.16 (2H, d, J=6 Hz),3.63 (2H, d, J=7 Hz), 5.08 (2H, s), 6.23 (1H, t, J=7 Hz) and 7.2 (5H,s).

DESCRIPTION 86-Methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-2(and3)-enoic acids, benzyl esters (D8) ##STR24##

The benzyl ester (D7) (26.46 g, 0.096 mole) in dry acetonitrile (500 ml)under N₂ was cooled to 0° in an ice bath and treated with1,1'-carbonyldiimidazole (15.69 g, 0.096 mole) in one portion. After 1 hat 0°, a solutiion of O-methyl-L-tyrosine N-methylamide (20 g, 0.096mole) in dry acetonitrile (200 ml) was added dropwise with stirring. Oncompleting the addition the mixture was maintained at 0° for 1 h andthen allowed to warm to room temperature. Stirring was continuedovernight and then solvent was removed in vacuo and the residue taken upin ethyl acetate. After washing with 1N HCl (2×200 ml) and extractingthe combined aqueous layers with ethyl acetate (3×100 ml), the combinedorganic layers were washed with water (100 ml), dried with anhydrousMgSO₄, filtered and evaporated to dryness to give a red oil.Purification by chromatography on silica gel with chloroform followed by2% methanol/chloroform gave the title compounds as a yellow gum (41.01g, 92%). Recrystallization of a sample from ethyl acetate/pentane gave awhite crystalline product, m.p. 98°-101° (Found: C, 69.38; H, 7.50; N,5.73. C₂₇ H₃₄ N₂ O₅ requires C, 69.51; H, 7.34; N, 6.00%).

DESCRIPTION 9 4-Methylaminocarbonyl-2-(2-methylpropyl)but-2-enoic acid(D9) ##STR25##

A slow stream Of anhydrous methylamine was passed into a solution of2-(2-methylpropyl)pent-2-ene dicarboxylic anhydride (1.3 g) in dry ether(25 ml) at 0° C. After 1 h the methylamine source was removed and thesolution was evaporated to dryness in vacuo. The residue was dissolvedin ethyl acetate and extracted with 10% aqueous sodium carbonate. Theaqueous phase was acidified with dilute hydrochloric acid andre-extracted with ethyl acetate. The extracts were washed with water,dried (MgSO₄), and evaporated to dryness to leave the title compound asa white solid (0.75 g, 49%), m.p. 120°-122° C., after trituration withether; (Found: C, 60.4; H, 8.5; N, 6.95. C₁₀ H₁₇ NO₃ requires C, 60.3;H, 8.6; N, 7.05%).

δ (d₆ -DMSO): 0.85 (6H, d, J=8 Hz), 1.75 (1H, m), 2.15 (2H, d, J=6 Hz),2.7 (3H, d, J=5 Hz), 3.3 (2H, d, J=8 Hz), 6.05 (1H, t, J=8 Hz), 7.8 (1H,brs) and 12.4 (1H, brs).

DESCRIPTION 10 4-Methylaminocarbonyl-2-(2-methylpropyl)but-2-enoic acid,tert-butyl ester (D10) ##STR26##

A suspension of 4-methylaminocarbonyl-2-(2-methylpropyl)but-2-enoic acid(2.4 g) in chloroform (35 ml) was treated with an excess of isobutylene(30 ml) and concentrated sulphuric acid (3 drops) was added as catalyst.The mixture was stirred at room temperature in a sealed vessel for 8days. The product was purified by silica gel chromatography[chloroform(2.5 g, 81%), m.p. 35°-36° C. (pentane).

δ (CDCl₃): 0.85 (6H, d, J=8 Hz), 1.5 (9H, s), 1.7 (1H, m), 2.1 (2H, d,J=8 Hz), 2.8 (2H, d, J=6 Hz), 3.2 (2H, d, J=10Hz), 5.9 (1H, t, J=10Hz)and 6.5 (1H, brs).

DESCRIPTION 112-(1-Benzoylmercapto-2-[methylaminocarbonyl]ethyl)-4-methylpentanoicacid, tert-butyl ester (D11) ##STR27##

A mixture of 4-methylaminocarbonyl-2-(2-methylpropyl)-but-2-enoic acid,tert-butyl ester (1.25 g, 4.9 mmol) and thiobenzoic acid (1.3 g, 9.4mmol) was heated at 80° C. under nitrogen for 48 h. The product waschromatographed on neutral alumina (50 g) using chloroform as the eluantto give a foam (0.5 g) containing the title compound as the majorconstituent.

δ (CDCl₃): 0.9 (m), 1.45 (m), 1.65 (m), 2.6 (m), 2.8 (d, J=5 Hz), 2.82(d, J=5 Hz), 3.0 (m), 3.2 (m), 4.3 (m), 5.9 (m), 7.4 (m), 7.55 (m) and7.95 (m). Observed FAB (M+H)+394. C₂₁ H₃₁ NO₄ S requires M 393.

DESCRIPTION 12 6-Methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-phenylethyl]-amino]carbonyl]hept-2(and 3)-enoic acid, methyl ester (D12)##STR28##

Prepared from 4-methoxycarbonyl-2-(2-methylpropyl)but-2-enoic acid (2.0g, 10 mmol) and L-phenylalanine-N-methylamide (1.87 g, 10.5 mmol) asdescribed for Description 8. The product was chromatographed on silicagel with methanol-dichloromethane (1:24) as the eluant to give the titlecompound (2.5 g, 69%) as a foam m.p. 78°-83° C. (Found: C, 66.7; H, 8.2;N, 7.6. C₂₀ H₂₈ N₂ O₄ requires C, 66.65; H, 7.85; N, 7.75%).

DESCRIPTION 136-Methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-3-enoicacid (D13) ##STR29##

A solution of the methyl ester (D6; 7.2 g) in methanol (80 ml) wastreated with sodium hydroxide (1.1 g) in water (20 ml) and the solutionwas stirred at room temperature overnight. The solution was diluted withwater, the methanol was removed in vacuo and the aqueous solution waswashed with ethyl acetate. The aqueous solution was acidified with 5NHCland extracted

with ethyl acetate (3×50 ml). The combined organic extracts were dried(Na₂ SO₄), filtered, and the solvent evaporated in vacuo to give thetitle compound as a foam (6.6 g).

DESCRIPTION 146-Methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-2(and3)-enoic acid, tert-butyl ester (D14) ##STR30##

A solution of6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-3-enoicacid (D13; 6.6 g) in dichloromethane (50 ml) and excess of isobutylenewere placed in a sealed vessel with concentrated sulphuric acid for 7days. After washing with 10% sodium carbonate solution, followed bywater, the organic extracts were dried (Na₂ SO₄), filtered andevaporated to dryness to give a red oil (4.1 g). Column chromatographyon silica gel, eluting with 10% methanol/chloroform gave the titlecompound as a foam (3.2 g).

(Found: C,66.51; H,8.31; N,6.37. C₂₄ H₃₆ N₂ O₅ requires: C,66.64;H,8.39; N,6.48%).

Observed FAB (M+H)⁺ 433 C₂₄ H₃₆ N₂ O₅ requires M 432.

DESCRIPTION 156-Methyl-4-[[[2-(R)-benzyloxy-1-(S)-[(methylamino)carbonyl]propyl]amino]carbonyl]hept-2(and3)-enoic acids, methyl esters (D15) ##STR31##

The methyl ester (D5; 3.1 g) in dry acetonitrile (75 ml) under N₂, wascooled to 0° in an ice bath and treated with 1,1'-carbonyldiimidazole(2.74 g) in one portion. After 1 h at 0°, a solution ofO-benzyl-L-threonine N-methylamide (3.44 g) in dry acetonitrile (25 ml)was added dropwise. The solution was stirred at room temperatureovernight, the solvent removed in vacuo and the residue taken up inethyl acetate. After washing with 1N HCl (2×25 ml) and extracting thecombined aqueous layers with ethyl acetate (3×50 ml), the combinedorganic layers were washed with water (50 ml), dried (Na₂ SO₄), filteredand evaporated to dryness to give an oil. Purification by chromatographyon silica gel, eluting with chloroform, followed by 5%methanol/chloroform gave the title compounds as a foam (4.4 g).

(Found: C,65.51; H,8.03; N,6.80. C₂₂ H₃₂ N₂ O₅ requires C,65.32; H,7.97;N,6.92%).

DESCRIPTION 166-Methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(3-indolyl)ethyl]amino]carbonyl]hept-2(and3)-enoic acid, methyl ester (D16) ##STR32##

Prepared from 4-methoxycarbonyl-2-(2-methylpropyl) but-2-enoic acid(1.75 g, 8.75 mmol) and L-tryptophan-N-methylamide (2.64 g, 13 mmol) asdescribed for Description 8. The product was chromatographed on silicagel with methanol-chloroform (1:99) as the eluant to give the titlecompound (2.8 g, 80%) as a foam m.p. 60°-65° C.

21 (Found: C,66.07; H,7.37; N,10.53. C₂₂ H₂₉ N₃ O₄ requires C,66.14;H,7.32; N,10.52%).

EXAMPLE 13-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, methyl ester (El) ##STR33##

A solution of 6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-3-enoic acid, methyl ester(D6; 500 mg, 1.3 mmol) in thiolacetic acid (3 ml) was stirred at roomtemperature for 14 days, then was evaporated to dryness in vacuo. Columnchromatography (30 g silica) of the residue, eluting with 10 to 25%ethyl acetate/dichloromethane gave the title compound (100 mg, 17%) m.p.118°-135° C. Observed mass 466.2137; C₂₃ H₂₄ N₂ O₆ S requires 466.2136Preparative HPLC (Lichrosorb diol), eluting with 20/80 (1%methanol/dichloromethane)/hexane gave two single isomers of the titlecompound.

Isomer A, mp 162°-165° C. had the longer retention time.

δ (CDCl₃): 0.85 (3H, d, J=7 Hz), 0.87 (3H, d, J=7 Hz), 1.2-1.7 (m), 2.31(3H, s), 2.45 (1H, dd, J=8, 17 Hz), 2.63 (1H, dd, J=5, 17 Hz), 2.6 (1H,m), 2.71 (3H, d, J=5 Hz), 2.95 (1H, dd, J=7, 14 Hz), 3.03 (1H, dd, J=7,14 Hz), 3.78 (3H, s), 3.66 (3H, s), 3.89 (1H, m), 4.55 (1H, q, J=8 Hz),5.58 (1H, brd), 6.33 (1H, d, J=8 Hz), 6.83 (2H, d, J=9 Hz) and 7.14 (2H,d, J=9 Hz).

Isomer B, mp 116°-118° C. had the shorter retention time.

δ (CDCl₃): 0.84 (3H, d, J=7 Hz), 0.86 (3H, d, J=7 Hz), 1.2-1.7 (m), 2.32(3H, s), 2.49 (2H, d, J=7 Hz), 2.7 (1H, m), 2.72 (3H, d, J=5 Hz), 2.97(1H, dd, J=7, 14 Hz), 3.04 (1H, dd, J=7, 14 Hz), 3.78 (3H, s), 3.66 (3H,s), 3.97 (1H, m), 4.52 (1H, q, J=8 Hz), 5.67 (1H, brs), 6.43 (1H, d, J=8Hz), 6.83 (2H, d, J=9 Hz) and 7.15 (2H, d, J=9 Hz).

EXAMPLE 23-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid methyl ester (E2) ##STR34##

An ice-cooled solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, methyl ester (El; 100 mg, 0.21 mmol) in nitrogen-purged methanol(10 ml) was treated with 35% aqueous ammonia (0.5 ml), and the reactionmixture was stirred under nitrogen for 20 h. The solution was evaporatedin vacuo, then was triturated with water, filtered and dried to give thetitle compound (46 mg, 52%), m.p. 80°-86° C. Observed mass 424.2028; C₂₁H₃₂ O₅ N₂ S requires 424.2033.

Each of the separated diastereoisomers of the S-acetyl compound ofExample 1 (5-15 mg) were individually dissolved in nitrogen-purgedmethanol (1 ml) and treated with 35% aqueous ammonia (0.1 ml). Thesolutions were stirred at room temperature under nitrogen for 2 h, thenwere evaporated in vacuo, and the residues were triturated with waterand dried to give:

Isomer A, m.p. 193°-196° C:

δ (CDl₃): 0.87 (6H, d, J=6 Hz), 1.2-1.7 (3H, m), 1.67 (1H, d, J=9 Hz),2.32 (1H, dd, J=10, 15 Hz), 2.41 (1H, m), 2.59 (1H, dd, J=4, 15 Hz),2.73 (3H, d, J=5 Hz), 3.0 (2H, d, J=8 Hz), 3.21 (1H, m), 3.70 (3H, s),3.78 (3H, s), 4.59 (1H, q, J=7 Hz), 5.66 (1H, brd), 6.34 (1H, d, J=8Hz), 6.83 (2H, d, J=9 Hz) and 7.13 (2H, d, J=9 Hz).

Observed mass. 424.2039; C₂₁ H₃₂ N₂ O₅ S requires 424.2031 and Isomer B:

δ (CDl₃): 0.86 (6H, t, J=7 Hz), 1.2-1.7 (3H, m), 2.00 (1H, d, J=9 Hz),2.49 (1H, m), 2.51 (1H, dd, J=9, 15 Hz), 2.68 (1H, dd, J=5, 16 Hz), 2.73(3H, d, J=5 Hz), 2.97 (1H, dd, J=7, 13 Hz), 3.07 (1H, dd, J=7H, 13 Hz),3.28 (1H, m), 3.72 (3H, s), 3.79 (3H, s), 4.55 (1H, q, J=7 Hz), 5.64(1H, brs), 6.33 (1H, d, J=7 Hz), 6.84 (2H, d, J=8 Hz) and 7.14 (2H, d,J=7 Hz).

Observed mass. 424.2024; C21H32N205S requires 424.2031.

EXAMPLE 33-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, benzyl ester (E3) ##STR35##

The benzyl ester (D8) (41 g, 0.088 mole) was dissolved in thiolaceticacid (170 ml) and left to stand at room temperature under nitrogen for19 days. Volatile material was removed in vacuo and the residuechromatographed on silica gel with initially ether, rising slowly toethyl acetate. After removal of high R_(f) impurities the title compoundwas obtained as a mixture of diastereomers as a pale yellow foam (29.76g, 62.4%). (Found: C, 64.29; H, 7.16; N, 4.86. C₂₉ H₃₈ N₂ SO₆ requiresC, 64.18; H, 7.06; N, 5.16%). Later fractions contained olefinicmaterial (D8). However, more complete reaction can be obtained byaddition of a suitable base, for example triethylamine. An aliquot (2ml) was removed from the above reaction mixture after 14 days, treatedwith triethylamine (3 drops) and left to stand for a further 5 days atroom temperature under nitrogen. The same work-up procedure as for thebulk of the reaction mixture described above gave only the diastereomermixture (E3) with no olefinic material remaining.3-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, benzyl ester (E4, Isomer A, 2.0 g 0.004 mol) was dissolved inchloroform (100 ml) under an atmosphere of nitrogen. The reactionmixture was cooled to 0° C. and acetic anhydride (2.04 g, 0.02 mol) wasadded followed by N-methylmorpholine (2.02 g, 0.02 mol). The reactionmixture was allowed to warm to room temperature, then stirred undernitrogen for 3 days.

The chloroform solution was washed with 10% aqueous citric acid solutionthen water, then dried (MgSO₄) and concentrated in vacuo to give a whitesolid which was recrystallised from ether to give the title compound asa single isomer (Isomer A) (1.4 g), m.p. 89°-90° C. (Found: C,63.94;H,7.25; N,5.13. C₂₉ H₃₈ N₂ O₆ S requires C,64.18; H,7.06; N,5.16%).

δ (CDl₃): 0.84(6H,t,J=7 Hz), 1.17-1.71(3H,m), 2.27(3H, s),2.45-2.7(3H,m), 2.7(3H,d,J=5 Hz), 2.85-3.04(2H,m), 3.78(3H,s),3.92(1H,m), 4.52(1H,q,J=8 Hz), 5.12(2H,s), 5.52(1H,brd), 6.32(1H,d,J=8Hz), 6.79(2H,d,J=9 Hz), 7.12(2H,d,J=9 Hz) and 7.36(5H,m).

EXAMPLE 43-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, benzyl ester (E4) ##STR36##

An ice-cooled solution Of3-acetylmercapto-6-methyl-4[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-amino]carbonyl]heptanoic acid, benzyl ester (E3; 1 g, 2 mmol) innitrogen-purged methanol (100 ml) was treated with 35% aqueous ammonia(10 ml). The mixture was stirred at room temperature under nitrogen for3 h and the precipitated solid (100 mg) was filtered off. The filtratewas evaporated in vacuo, and the residue was crystallised from methanol(5 ml) and ether (120 ml), to give a single isomer (isomer A) of thetitle compound (300 mg), m.p. 199°-201° C.

(Found C,64.77; H,7.31; N,5.68. C₂₇ H₃₆ N₂ O₅ S requires C,64.77;H,7.25; N,5.60%).

δ (CDl₃): 0.84 (3H, d, J=7 Hz), 0.86 (3H, d, J=7 Hz), 1.3-1.7 (3H,m),1.66 (1H, d, J=9 Hz), 2.38 (1H, dd, J=9, 15 Hz), 2.41 (1H, m), 2.66 (1H,dd, J=4, 15 Hz), 2.73 (3H, d, J=5 Hz), 2.97 (2H, d, J=8 Hz), 3.22 (1H,m), 3.74 (3H, s), 4.57 (1H, q, J=8 Hz), 5.14 (2H, s), 5.63 (1H, brd),6.33 (1H, d, J=8 Hz), 6.81 (2H, d, J=9 Hz), 7.12 (2H, d, J=9 Hz) and7.36 (5H, m).

Column chromatography of the mother liquors on silica, eluting with 3:1ether:chloroform gave two further single diastereoisomers. Isomer B, theslower running isomer was obtained as a foam, which solidified onstanding, mp 133°-135° C. (Found: C,64.66; H,7.41; N,5.58. C₂₇ H₃₆ N₂ O₅S requires C,64.77; H,7.25; N,5.60%).

δ (CDl₃): 0.73 (3H, d, J=7 Hz), 0.79 (3H, d, J=7 Hz), 1.2-1.7 (3H, m),1.90 (1H, d, J=9 Hz), 2.36 (1H, m), 2.64 (1H, dd, J=9, 17 Hz), 2.69 (3H,d, J=6 Hz), 2.81 (1H, dd, J=4, 17 Hz), 2.94 (1H, dd, J=7, 16 Hz), 3.03(1H, dd, J=7, 16 Hz), 3.26 (1H, m), 3.78 (3H, s), 4.59 (1H, q, J=7 Hz),5.15 (2H, m), 5.98 (1H, brd), 6.16 (1H, d, J=8 Hz), 6.82 (2H, d, J=9Hz), 7.10 (2H, d, J=9 Hz) and 7.36 (5H, m).

Isomer C, the faster running isomer was obtained as a gum.

δ (CDl₃): 0.81 (3H, d, J=6 Hz), 0.83 (3H, d, J=6 Hz), 1.2-1.7 (3H, m),1.99 (1H, d, J=9 Hz), 2.47 (1H, m), 2.56 (1H, dd, J=8, 15 Hz), 2.72 (1H,m), 2.72 (3H, d, J=6 Hz), 2.96 (1H, dd, J=7, 15 Hz), 3.04 (1H, dd, J=7,15 Hz), 3.29 (1H, m), 3.77 (3H, s), 4.55 (1H, q, J=7 Hz), 5.16 (2H, s),5.75 (1H, brd), 6.38 (1H, d, J=8 Hz), 6.81 (2H, d, J=9 Hz), 7.12 (2H, d,J=9 Hz) and 7.36 (5H, m).

Isomer D was obtained from a mixture of isomers B, and D by preparativeHPLC (Lichrosorb diol), eluting with 18/82 (1%methanol/dichloromethane)/hexane.

δ (CDl₃): 0.68 (3H, dd, J=7 Hz), 0.78 (3H, d, J=7 Hz), 1.1-1.6 (3H, m),2.09 (1H, d, J=10 Hz), 2.36 (1H, m), 2.63 (1H, dd, J=6,17 Hz), 2.74 (3H,d, J=6 Hz), 2.85 (1H, dd, J=5,17 Hz), 2.99 (1H, dd, J=7,16 Hz), 3.09(1H, dd, J=7,16 Hz), 3.29 (1H, m), 3.79 (3H, s), 4.68 (1H, q, J=7 Hz),5.91 (1H, d, J=8 Hz), 6.24 (1H, brd), 6.82 (2H, d, J=9 Hz) and 7.10 (2H,d, J=9 Hz).

EXAMPLE 5 3-3-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid (E5) ##STR37##

A solution of3-acetylmercapto-6-methyl-4-[[[1-(S)[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, benzyl ester (E3; 300 mg, 0.55 mmol) in 4.5% formic acid/methanol(6 ml) was added, under nitrogen, to a stirred suspension of palladiumblack (310 mg) in the same solvent (15 ml). After 90 min the mixture wasfiltered through kieselguhr and evaporated in vacuo. Columnchromatography using reverse phase silica (30 g) and eluting with 25%aqueous methanol gave two single diastereoisomers of the title compound,as monohydrates.

Isomer A, m.p. 92°-95° C. (54 mg).

(Found: C, 56.34; H. 7.11; N, 6.08. C₂₂ H₃₂ N₂ O₆ S.H₂ O requires C,56.15; H, 7.28; N, 5.95%).

δ (CDl₃): 0.85 (3H, d, J=6 Hz), 0.91 (3H, d, J=6 Hz), 1.2-1.7 (3H, m),2.31 (3H, s), 2.50 (1H, dd, J=3, 17 Hz), 2.67 (1H, dd, J=7, 17 Hz), 2.68(3H, d, J=5 Hz), 2.85-3.0 (2H, m), 3.15 (1H, t, J=10 Hz), 3.74 (3H, s),3.80 (1H, m), 4.80 (1H, q, J=8 Hz), 6.36 (1H, m), 6.77 (2H, d, J=9 Hz),7.07 (2H, d, J=9 Hz) and 8.41 (1H, d, J=10Hz). and

Isomer B, m.p. 106°-110° C. (70 mg). (Found: C, 56.33; H, 7.17; N, 6.15.C₂₂ H₃₂ N₂ O₆ S.H₂ O requires C, 56.15; H, 7.28; N, 5.95%). δ (CDCl₃):0.77 (3H, s), 0.86 (3H, s), 1.1-1.7 (3H, m), 2.34 (3H, s), 2.73 (2H, d,J=4 Hz), 2.82 (3H, d, J=4 Hz), 2.8-3.0 (3H, m), 3.75 (3H, s), 3.88 (1H,brd, J=11 Hz), 4.80 (1H, q, J=8 Hz), 6.61 (1H, m), 6.78 (2H, d, J=9 Hz),7.11 (2H, d, J=9 Hz) and 8.53 (1H, d, J=9 Hz).

Similar treatment of the single isomer of the acetylmercapto compound(E3, Isomer A; 1.4 g, 2.6 mmol) gave the title compound as a singleisomer (Isomer A), 980 mg (84%).

EXAMPLE 63-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid (E6) ##STR38##

Each of the separate isomers of the S-acetyl compound of Example 5(27-42 mg) were individually dissolved in nitrogen-purged methanol (4ml) and treated with 35% aqueous ammonia (0.4 ml). The solutions werestirred at room temperature under nitrogen for 6 h, then were evaporatedin vacuo, and the residues were triturated with ether to give:

Isomer A, m.p. 171°-176° C.: δ(CDCl₃) 0.86 (6H, t, J=6 Hz), 1.3-1.7 (3H,m), 1.79 (1H, d, J=9 Hz), 2.43 (1H, dd, J=5, 15 Hz), 2.66 (1H, dd, J=4,15 Hz), 2.72 (3H, d, J=5 Hz), 2.8-3.1 (4H, m), 3.74 (3H, s), 4.73 (1H,q, J=7 Hz), 6.29 (1H, brs), 6.89 (2H, d, J=9 Hz), 7.09 (2H, d, J=9 Hz)and 8.02 (1H, brd, J=9 Hz). and

Isomer B, m.p. 107°-110° C.: δ (CDCl₃) 0.76 (3H, d, J=7 Hz), 0.79 (3H,d, J=7 Hz) 2.34 (1H, d, J=10 Hz), 2.73 (2H, d, J=6 Hz), 2.80 (3H, d, J=6Hz), 2.85-3.2 (4H, m), 3.77 (3H, s), 4.71 (1H, q, J=8 Hz), 6.26 (1H,brs), 6.79 (2H, d, J=9 Hz), 7.09 (2H, J=9 Hz) and 7.94 (1H, brd, J=9Hz).

EXAMPLE 7 3-Benzoylmercapto-N⁵ -methyl-N¹ -[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E7) ##STR39##

2-(1-Benzoylmercapto-2-[methylaminocarbonyl]ethyl)-4-methylpentanoicacid, tert-butyl ester (D11; 0.5 g) was treated with trifluoroaceticacid (10 ml) at 0° C. for 2h and evaporated to dryness in vacuo to give2-(1-benzoylmercapto-2-[methylaminocarbonyl]ethyl)4-methylpentanoicacid.

[δ(CDCl₃) no signal at 1.5 ppm indicating absence of a tert-butyl group]

N,N-Dicyclohexylcarbodiimide (235 mg) and hydroxybenzotriazole (145 mg)was added to an ice-cooled solution of the above acid andO-methyl-L-tyrosine-N-methylamide (220 mg) in dry dichloromethane (20ml), and the mixture was stirred at room temperature for 18 h. Theprecipitated solid was filtered off, and washed with a littledichloromethane, then the organic solution was washed successively with1N hydrochloric acid, IN sodium bicarbonate and water. The solution wasdried (MgSO₄) and evaporated to dryness in vacuo. The residual solid waschromatographed on neutral alumina using chloroform-pentane (1:1) aseluant. The solid product was triturated with ether to give the titlecompound (130 mg), m.p. 140°-144° C. as a mixture of diastereoisomers.

Observed M⁺ 527.2449. C₂₈ H₃₇ N₃ O₅ S requires M 527.2453.

EXAMPLE 8 3-Mercapto-N⁵ -methyl-N¹-[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E8) ##STR40##

An ice-cooled solution of 3-benzoylmercapto-N⁵ -methyl-N¹-[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl-2-(2-methylpropyl)pentanediamide(E7; 40 mg) in nitrogen-purged methanol (5 ml) was treated with 35%aqueous ammonia (2.5 ml) and the reaction mixture was stirred undernitrogen for 2 h. The solution was filtered and the filtrate wasevaporated to dryness in vacuo The residue was triturated with ether,filtered, and dried to give the title compound (20 mg, 62%), m.p175°-190° C. as a mixture of diastereoisomers. Observed M⁺ 423.2190. C₂₁H₃₃ N₃ O₄ S requires M 423.2191.

Similar treatment of 3-acetylmercapto-N⁵ -methyl-N¹-(1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)-ethyl2-(2-methylpropyl)pentanediamide(Ell, Isomer A; 48 mg, 0.1 mmol) gave the title compound as a singleisomer

(Isomer A), (28 mg, 64%), mp 243°-250°C. (Found: C,59.89; H,7.86;N,9.55. C₂₁ H₃₃ N₃ O₄ S requires C,59.55; H,7.85; N,9.92%). δ (CDCl₃+drop d₆ -DMSO): 0.83(3H,d,J=8 Hz), 0.85(3H,d, J=8 Hz), 1.2-1.6(3H,m),1.74(1H,d,J=7 Hz), 2.0(1H,dd, J=11,13 Hz), 2.10(1H,dd,J=3,13 Hz),2.65(6H,d,J=5 Hz), 2.79(1H,dd,J=9,13 Hz), 2.98(1H,dd,J=5,13 Hz),3.73(3H,s), 4.56(1H,m), 6.87(2H,d,J=9 Hz), 7.15(2H,d,J=9 Hz), 7.55(2H,m) and 7.98(1H,d,J=9 Hz).

EXAMPLE 93-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-phenylethyl]amino]carbonyl]-heptanoicacid, methyl ester (E9) ##STR41##

A solution of6-methyl-4-[[[1-(S)-[(methylamino)-carbonyl]-2-phenylethyl]amino]carbonyl]-hept-3-enoicacid, methyl ester (D12; 2.3 g, 6.3 mmol) in thiolacetic acid (20 ml)was stirred at room temperature for 14 days, then was evaporated todryness in vacuo. The product was chromatographed on silica gel (100 g)eluting with 10 to 25% ethyl acetate/pentane, to give the title compound(1.35 g, 49%) (mixture of diastereoisomers).

The product was recrystallised (×2) from ethyl acetate

to give a solid (0.7 g) m.p. 188°-189° C. which was shown by NMR to be amixture of two diastereoisomers (ratio 3:1).

(Found: C, 60.9; H, 7.3; N, 6.35. C₂₂ H₃₂ N₂ O₅ S requires C, 60.55; H,7.45; N, 6.4%). δ(CDCl₃) 0.85 (6H, m), 1.2-1.7 (3H, m), 2.3 (s) and 2.32(s) (total 3H), 2.4 (1H, m), 2.6 (2H, m), 2.71 (3H, d, J=6 Hz), 3.07(2H, m), 3.66 (s) and 3.68 (s) (ratio 3:1 - total 3H), 3.9 (1H, m), 4.65(1H, m), 5.78 (brd) and 5.83 (brd) (ratio 3:1 - total 1H), 6.43 (d, J=8Hz) and 6.51 (d, J=8 Hz) (ratio 3:1 - total 1H) and 7.25 (5H, m).

EXAMPLE 103-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-phenylethyl]amino]carbonyl]heptanoicacid, methyl ester (E10) ##STR42##

An ice-cooled solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-phenylethyl]amino]carbonyl]heptanoicacid, methyl ester, m.p. 188°-189° C., (E9; 100 mg) in nitrogen-purgedmethanol (5 ml) was treated with 35% aqueous ammonia (2 ml) and thereaction mixture was stirred under nitrogen for 3.5 h. The solution wasfiltered, and the filtrate was evaporated to dryness in vacuo. Theresidue was triturated with water and the solid was collected andrecrystallised from ethyl acetate-ether to give the title compound (25mg), m.p. 169°-171° C., as a single diastereoisomer (Isomer A).

(Found: C, 60.65; H, 7.85; N, 7.05. C₂₀ H₃₀ N₂ O₄ S requires C, 60.9; H,7.65; N, 7.1%). δ(CDCl₃): 0.85 (6H, d, J=5 Hz), 1.2-1.7 (3H, m), 1.64(1H, d, J=9 Hz), 2.3 (1H, dd, J=9, 16 Hz), 2.42 (1H, m), 2.58 (1H, dd,J=4, 16 Hz), 3.07 (1H, d, J=8 Hz), 3.2 (1H, m), 3.68 (3H, s), 4.65 (1H,q, J=8 Hz), 5.75 (1H, brd), 6.39 (1H, d, J=8 Hz) and 7.25 (5H, m).

EXAMPLE 11 3-Acetylmercapto-N⁵ -methyl-N¹-[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E11) ##STR43##

Diisopropylethylamine (0.13 ml, 0.7 mmol) and ethyl chloroformate (0.07ml, 0.7 mmol) were added to a solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid (E5, Isomer A; 300 mg, 0.66 mmol) in dry THF (10 ml), at -20° C.After 15 min a solution of methylamine (20 mg) in THF (0.8 ml) was addeddropwise and the mixture was stirred in an ice bath for 3 h. The solventwas evaporated in vacuo, and a solution of the residue indichloromethane was washed successively with water, 1M hydrochloricacid, water, saturated sodium hydrogen carbonate and brine. The solutionwas dried (MgSO₄) and evaporated in vacuo. Column chromatography onsilica (10 g), eluting with 5% methanol/ethyl acetate gave the titlecompound, mp 195°-199° C.

(Found C,58.97; H,7.78; N,.8.92. C₂₃ H₃₅ N₃ O₅ S requires C,59.33;H,7.58; N,9.03%).

δ(CDCl₃): 0.84(6H,q,J=3 Hz), 1.2-1.7(3H,m), 2.10(1H,dd, J=7,15 Hz),2.30(3H,s), 2.31(1H,dd,J=5,15 Hz), 2.68(1H, m), 2.74(3H,d,J=5 Hz),2.76(3H,d,J=5 Hz), 2.95(1H,dd,J=6,14 Hz), 3.08(1H,dd,J=6,14 Hz), 3.78(3H,s), 3.83(1H,m), 4.63(1H,q,J=7 Hz), 5.49(1H,brs), 5.88(1H,brs),6.68(1H, d,J=7 Hz), 6.82(2H,d,J=8 Hz) and 7.14(2H,d,J=8 Hz).

EXAMPLE 122-[3-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid, t-butyl ester (E12). ##STR44##

A solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-amino]carbonyl]heptanoicacid (E5, Isomer A; 90 mg, 0.2 mmol),N-ethyl-N'-dimethylaminopropylcarbodiimide hydrochloride (42 mg, 0.22mmol), 1-hydroxybenzotriazole (34 mg, 0.22 mmol), t-butylglycinehydrochloride (37 mg, 0.22 mmol) and diisopropylethylamine (0.07 ml, 0.4mmol) in dichloromethane (2 ml), was stirred at room temperature for 18h. The mixture was diluted with dichloromethane and was washedsuccessively with saturated sodium hydrogen carbonate, water, 1M citricacid and brine. The solution was dried (NaSO₄) and evaporated in vacuoto leave the title compound as a single isomer (Isomer A) (74 mg, 68%),mp 192°-194° C. (ethyl acetate).

(Found: C,59.51; H,7.70; N,7.33. C₂₈ H₄₃ N₃ O₇ S requires C,59.45;H,7.66; N,7.43%).

δ(CDCl₃): 0.77(3H,d,J=6 Hz), 0.83(3H,d,J=6 Hz), 1.2-1.7 (3H,m),1.47(9H,s), 2.30(3H,s), 2.35(2H,m), 2.56(1H, m), 2.77(3H,d,J=5 Hz),3.00(1H,dd,J=10,15 Hz), 3.10(1H,dd,J=7,15 Hz), 3.70(1H,dd,J=6,19 Hz),3.78(1H,s), 3.95(1H,dd,J=6,19 Hz), 4.00(1H,m), 4.64(1H,q,J=7 Hz),6.05(1H,m), 6.33(1H,brt), 6.83(2H,d,J=9 Hz), 7.14(2H,d,J=9 Hz) and7.43(1H,d,J=7 Hz).

EXAMPLE 13 2-[3-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid, t-butyl ester (E13) ##STR45##

An ice-cooled solution of 2-[3-acetylmercapto-6-met4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoic acid, t-butyl ester (E12,Isomer A; 30 mg, 0.053 mmol) in nitrogen-purged methanol (4 ml) wastreated with 35% aqueous ammonia (0.3 ml), under nitrogen, and wasstirred for 2 h at room temperature. The solvents were evaporated invacuo and the residue was triturated with hexane to give the titlecompound as a single isomer (Isomer A), mp 223°-226° C.

(Found: C,59.52; H,7.68; N,7.88. C₂₆ H₄₁ N₃ O₆ S requires C,59.63;H,7.89; N,8.02%).

δ(CDCl₃): 0.84(6H,dd,J=6,8 Hz), 1.3-1.7(3H,m), 1.48 (9H,s),2.24(1H,d,J=10 Hz), 2.28(1H,m), 2.45(1H,dd,J=15, 5 Hz), 2.50(1H,m),2.76(3H,d,J=5 Hz), 2.99(1H,dd,J=15, 9 Hz), 3.09(1H,dd,J=15,7 Hz),3.12(1H,m), 3.77(3H,s), 3.89(1H,dd,J=5,7 Hz), 4.64(1H,q,J=8 Hz),5.98(1H,brd), 6.47(1H,brt), 6.84(2H,d,J=9 Hz), 7.15(2H,d,J=9 Hz) and7.18(1H,brs).

EXAMPLE 142-[3-Acetylmercapto-6-methyl-4-[[[1-(S)-(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid (E14) ##STR46##

A solution of2-[3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid, t-butyl ester (E12, Isomer A; 60 mg, 0.11 mmol) in trifluoroaceticacid (1.5 ml) and water (0.05 ml) was stirred in an ice bath for 1 h,then at room temperature for 2 h. The solvent was evaporated in vacuoand the residue was azeotroped dry with toluene to leave the titlecompound as a single isomer (Isomer A), mp 188°-192° C.

EXAMPLE 152-[3-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid (E15) ##STR47##

A solution of2-[3-acetylmercapto-6-methyl-4-[[[1-(S)[(methylino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylamino]ethanoicacid (E14, Isomer A; 15 mg, 0.029 mmol) in nitrogen-purged methanol (5ml) was cooled in ice and treated with 35% aqueous ammonia (0.3 ml).After 2 h the solvents were evaporated in vacuo and the residue wastriturated with ethyl acetate to give the title compound as a singleisomer (Isomer A), 11 mg (80%), mp 228°-231° C.

EXAMPLE 16 3-Acetylmercapto-N5-(aminocarbonyl)methyl-N¹-[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2methylpropyl)pentanediamide(E16) ##STR48##

A mixture of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid (E5, Isomer A; 226 mg, 0.5 mmol), N-ethyl-N¹-dimethylaminopropylcarbodiimide hydrochloride (116 mg, 0.6 mmol),1-hydroxybenzotriazole (90 mg, 0.58 mmol), glycinamide hydrochloride (64mg, 0.58 mmol) and diisopropylethylamine (0.2 ml, 1.14 mmol) indichloromethane was stirred at 5° C. for 2 h then at room temperatureovernight. The mixture was cooled in ice and the solid was collected byfiltration and washed well with dichloromethane. The solid wastriturated with 1M citric acid, then filtered, washed with water anddried. Recrystallisation from methanol/ether gave the title compound asa single isomer (Isomer A), 75 mg (30%) mp 218°-219° C.

δ(d₆ -DMSO): 0.78(6H,t,J=7 Hz), 1.1-1.5(3H,m), 2.1-2.3 (2H,m),2.24(1H,s), 2.55(3H,d,J=5 Hz), 2.74(1H,dd,J=10,14 Hz), 2.85(1H,dd,J=5,14Hz), 3.56(2H,d,J=6 Hz), 3.68(3H,s), 3.77(1H,m), 4.43(1H,m),6.81(2H,d,J=9 Hz), 7.10(2H,brd), 7.14(2H,d,J=9 Hz), 7.79(1H,brq),8.00(1H,t,J=6 Hz) and 8.12(1H,d,J=8 Hz).

EXAMPLE 17 N⁵ -(Aminocarbonyl)methyl-3-mercapto-N¹ -[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E17) ##STR49##

An ice-cooled solution of 3-acetylmercapto-N⁵ -(amino-carbonyl)methyl-N¹-[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E16, Isomer A; 44 mg, 0.087 mmol) in nitrogen-purged methanol (5 ml)was treated with 35% aqueous ammonia (0.4 ml) and was stirred at roomtemperature for 2.5h. The solvent was evaporated in vacuo and theresidue was triturated with ether to leave the title compound as asingle isomer (Isomer A), 28 mg (70%), mp 247°-252° C.

δ(d₆ -DMSO) 0.77(3H,d,J=6 Hz), 0.82(3H,d,J=6 Hz), 1.2-1.5(3H,m),2.04(2H,m), 2.30(1H,d,J=7 Hz), 2.33(1H,m), 2.57(3H,d,J=5 Hz),2.70(1H,d,J=10,14 Hz), 2.86(1H,dd,J=5,14 Hz), 3.08(1H,m), 3.62(2H,q,J=5Hz), 3.67(3H,s), 4.46(1H,m), 6.80(2H,d,J=9 Hz), 7.10(2H,brd),7.15(2H,d,J=9 Hz)), 7.81(1H,m), 8.06(1H,t,J=5 Hz) and 8.18(1H,d,J=8 Hz).

EXAMPLE 181-[3-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl]-4-methylpiperazine(E18) ##STR50##

A mixture of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]-carbonyl]heptanoicacid (E5, Isomer A; 226 mg, 0.5 mmol), N,N¹ -dicyclohexylcarbodiimide(103 mg, 0.5 mmol), 1-hydroxybenzotriazole (76 mg, 0.5 mmol) andN-methylpiperazine (0.05 ml, 0.5 mmol) in dichloromethane (8 ml) wasstirred at 5° C. for 1 h, then at room temperature overnight. Aftercooling, the precipitated solid was filtered off. The solution wasdiluted with dichloromethane and extracted with 0.25M citric acid. Theextracts were washed with dichloromethane then basified with 10% sodiumcarbonate, and extracted with ether. The ether extracts were washed withbrine, dried (Na₂ SO₄) and evaporated in vacuo. Trituration of the oilyresidue with ether, followed by recrystallisation of the resulting solidfrom ethyl acetate/hexane gave the title compound as a single isomer(Isomer A), 98 mg (37%) mp 133°-138° C.

(Found C,60.48; H,8.15; N,10.40. C₂₇ H₄₂ N₄ O₄ S requires C,60.65;H,7.92; N,10.48%).

δ(CDCl₃): 0.85(6H,q,J=3 Hz), 1.2-1.5(3H,m), 2.30(6H,s), 2.38(4H,brs),2.33-2.8(3H,m), 2.70(3H,d,J=5 Hz), 2.98 (2H,d,J=7 Hz), 3.4(2H,brs),3.5-3.7(2H,brm), 3.78(3H,s), 3.95(1H,q,J=6 Hz), 4.54(1H,q,J=7 Hz),5.71(1H,brd), 6.64(1H,brd), 6.82(2H,d,J=9 Hz) and 7.15(2H,d,J=9 Hz).

EXAMPLE 191-[3-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl]-4-methylpiperazine(E19) ##STR51##

A solution of1-[3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]-carbonyl]heptanoyl]-4-methylpiperazine(E18, Isomer A; 60 mg, 0.112 mmol) in methanol (5 ml) was purged withnitrogen and cooled in ice, then was treated with 35% aqueous ammonia(0.4 ml). After 6 h at room temperature, the solvents were evaporated invacuo and the residue was triturated with hexane to give the titlecompound as a single isomer (Isomer A), mp 65°-68° C.

δ(CDCl₃): 0.87(6H,d,J=6 Hz), 1.3-1.7(3H,m), 1.85(1H,d,J=8 Hz),2.35(7H,m), 2.42(1H,dd,J=8,16 Hz), 2.54(1H,m), 2.61(1H,dd,J=4,16 Hz),2.74(3H,d,J=5 Hz), 3.01(2H,d,J=8 Hz), 3.30(1H,m), 3.48(2H,brs),3.65(2H,brm), 3.79(3H,s), 4.58(1H,q,J=8 Hz), 5.35(1H,brm), 5.74(1H,brd),6.52(1H,d,J=8 Hz), 6.82(2H,d,J=9 Hz) and 7.15(2H,d,J=9 Hz).

EXAMPLE 201-[3-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl]morpholinehydrate (E20) ##STR52##

To a solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid (E5, Isomer A; 290 mg, 0.64 mmol) in acetonitrile (10 ml) at 0° C.was added 1,1'-carbonyl diimidazole (104 mg, 0.64 mmol) and the solutionwas stirred under nitrogen at 0° C. for 1 h. A solution of morpholine((56 mg, 0.64 mmol) in acetonitrile (2 ml) was then added dropwise, andthe solution was left at room temperature overnight. The mixture wasevaporated to dryness in vacuo and then dissolved in a mixture of ethylacetate and chloroform (4:1; 30 ml) and washed successively with sodiumcarbonate, 1M-hydrochloric acid and water. The organic fraction wasdried (MgSO₄) and chromatographed on silica gel 60 (100 g). Elution with5% methanol-ethyl acetate gave the title compound (70 mg) as a singleisomer (Isomer A), m.p. 146°-149° C., after trituration with ether.

(Found: C,57.73; H,7.64; N,7.59. C₂₆ H₃₉ N₃ O₆ S.H₂ O requires C,57.86;H,7.66; N,7.79%).

δ (CDCl₃) 0.86(d,J=Hz) and 0.87(d,J=5 Hz) (total 6H), 1.2-1.7(2H,m),2.32(3H,s), 2.48(1H,dd,J=4,16 Hz), 2.62(1H,dd,J=4,16 Hz),2.72(4H,brd,J=5 Hz), 2.98(2H,d,J=7 Hz), 3.3-3.75(8H,m), 3.76(3H,s),3.96(1H,q,J=6 Hz), 4.54(1H,q,J=6 Hz), 5.69(1H,brd), 6.61(1H,d,J=7 Hz),6.84(2H,d,J=8 Hz) and 7.16(2H,d,J=8 Hz).

EXAMPLE 211-[3-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl]morpholinehemihydrate (E21) ##STR53##

A solution of1-[3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)-carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl]morpholine(E20, Isomer A; 12 mg) in methanol (3 ml) was purged with nitrogen andcooled in ice. The solution was treated with 35% aqueous ammonia (1.5ml) at 0° C. for 0.5 h and then allowed to warm up to room temperature.After 1.5 h at room temperature the solution was evaporated to drynessin vacuo and the residue was washed twice with ether to give the titlecompound as a single isomer (Isomer A) m.p. 106°-108° C.

(Found: C,58.81; H,7.92; N,8.75. C₂₄ H₃₇ N₃ O₅ S 0.5H₂ O requiresC,58.99; H,7.83; N,8.60%).

δ (CDCl₃) 0.87(6H,d,J=7 Hz), 1.35-1.5(2H,m), 1.84(1H,d,J=9 Hz),2.40(1H,dd,J=8 Hz), 2.54(1H,m), 2.57(1H,dd,J=4,16 Hz), 2.72(3H,d,J=6Hz), 3.01((2H,d,J=7 Hz), 3.32(1H,m), 3.35-3.75(8H,m), 3.78(3H,s),4.58(1H,q,J=8 Hz), 5.77(1H,bd), 6.58(1H,d,J=8 Hz), 6.82(2H,d,J=8 Hz) and7.15(2H,d,J=8 Hz).

EXAMPLE 223-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, tert-butyl ester (E22) ##STR54##

A solution of6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]hept-2(and3)-enoic acid, tert-butyl ester (D14; 3 g) in thiolacetic acid (15 ml)was left at room temperature for 14 days, then evaporated to dryness invacuo. Column chromatography on silica gel, eluting with ethylacetate-pentane (1:1) gave the title compound (1.2 g) as a mixture ofisomers.

A single diastereoisomer (Isomer A), m.p. 147°-151° C. (fromether-pentane), was obtained by preparative HPLC (Lichrosorb diol),eluting with 15/85 (1% methanol/dichloromethane)/hexane.

(Found: C,61.08; H,7.94; N,5.53. C₂₆ H₄₄ ON₂ O₆ S requires C,61.39;H,7.93; N,5.51%).

δ (CDCl₃): 0.84(d,H=5 Hz) and 0.86(d,J=5 Hz) (total 6H), 1.2-1.7(2H,m),1.44(9H,s), 2.30(3H,s), 2.49(1H,dd,J=7,17 Hz), 2.58(1H,dd,J=5,17 Hz),2.60(1H,m), 2.70(3H,d,J=5 Hz), 2.93(1H,dd,J=8,14 Hz), 3.04(1H,dd,J=7,14Hz), 3.79(3H,s), 3.92(1H,q,J=6 Hz), 4.52(1H,q,J=7 Hz), 5.52(1H,brs),6.37(1H,d,J=8 Hz), 6.82(2H,d,J=8 Hz) and 7.15(2H,d,J=8 Hz).

EXAMPLE 233-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, tert-butyl ester (E23) ##STR55##

An ice-cooled solution of3-acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoicacid, tert-butyl ester (E22) (23 mg) in nitrogen-purged methanol (3 ml)was treated with 35% aqueous ammonia (1.5 ml) and the reaction mixturewas stirred under nitrogen for 3 h. The solution was filtered, and thefiltrate was evaporated to dryness in vacuo. The residue was partitionedbetween chloroform (5 ml) and water (2 ml). The organic layer was dried(MgSO₄) and evaporated to dryness in vacuo. The product wasrecrystallised from ether-hexane to give the title compound, (Isomer A),m.p. 133°-137° C.

(Found: C,61.72; H,8.45; N,5.90. C₂₄ H₃₈ N₂ O₅ S requires C,61.77;H,8.21; N,6.00%).

δ (CDCl₃): 0.86(6H,d,J=5 Hz), 1.3-1.75(2H,m), 1.46(9H,s), 1.65(1H,d,J=9Hz), 2.25(1H,dd,J=8,14 Hz), 2.44(1H,m), 2.55(1H,dd,J=4,14 Hz),2.72(3H,d,J=5 Hz), 3.02(2H,d,J=7 Hz), 3.20(1H,m), 3.77(3H,s),4.59(1H,q,J=7 Hz), 5.77(1H,brd), 6.44(1H,d,J=8 Hz), 6.82(2H,d,J=8 Hz)and 7.15(2H,d,J=8 Hz).

EXAMPLE 241-[3-Acetylmercapto-6-methyl-4-[[[2-(R)-benzyloxy-1-(S)-[(methylamino)carbonyl]propyl]amino]carbonyl]heptanoicacid, methyl ester (E24) ##STR56##

A solution of6-methyl-4-[[[2-(R)-benzyloxy-1-(S)-[(methylamino)carbonyl]propyl]amino]carbonyl]hept-2(and3)-enoic acid, methyl esters (D15; 4.4 g) in thiolacetic acid (25 ml)was left at room temperature for 21 days, then was evaporated to drynessin vacuo and chromatographed on silica gel (150 g). Elution with ethylacetate/pentane (1:1) gave the title compound as a 3:2 mixture ofdiastereoisomers (1.7 g) (Isomers A/B), m.p. 105°-106° C. (fromether-pentane).

(Found: C,59.95; H,7.74; N,5.84. C24H36N206S requires C,59.98; H,7.55;N,5.83%).

δ (CDCl₃) 0.90(m), 1.11(d,J=5 Hz) and 1.15(d,J=5 Hz) (ratio 3:2),1.33(m), 1.53(m), 1.68(m) 2.32(s), 2.6-2.85(m), 3.66(s) and 3.67(s)(ratio 3:2), 4.08(m), 4.55(brs), 4.67(s), 6.45(brs), 6.75(d,J=6 Hz),6.84(d,J=6 Hz) and 7.35(m). Further elution gave the title compound as a2:1 mixture of diastereoisomers (2.1 g) (Isomers C/D), m.p. 111°-113° C.(from ether-pentane).

(Found: C,60.35; H,7.25; N,5.84. C₂₄ H₃₆ N₂ O₆ S requires C,59.98;H,7.55; N,5.83%).

δ (CDC13): 0.88(m), 1.13(t,J=5 Hz), 1.32(m), 1.52(m), 1.62-1.80(m),2.33(s), 2.6-2.82(m), 3.64(s), 3.66(s), 4.0-4.5(m), 4.19-4.24(m),4.50(m), 4.58-4.64(m), 6.62(m), 6.79(m) and 7.3(m).

EXAMPLE 253-Mercapto-6-methyl-4-[[[2-(R)-benzyloxy-1-(S)-[(methylamino)carbonyl]propyl]amino]carbonyl]heptanoicacid, methyl ester (E23) ##STR57##

Each of the mixtures of diastereoisomers (A/B and C/D) of the S-acetylcompound of Example 24 were individually treated with 35% aqueousammonia in the usual manner, to give the title compound;

Isomer (A/B) (3:2 ratio of diastereoisomers), m.p. 113°-119° C. (from H₂O). (Found: C,59.04; H,7.88; N,6.26. C₂₂ H₃₄ N₂ O₅ S 0.5H₂ O requiresC,57.87; H,7.95; N,6.13%).

δ (CDCl₃): 0.94(m), 1.12(d,J=5 Hz), 1.13(d,J=5 Hz), 1.38(m), 1.5(m),1.71(mm), 1.90(0.6H,d,J=8 Hz), 2.06(0.4H,d,J=8 Hz), 2.57(m), 2.82(m),3.37(m), 3.72(s), 4.14(m), 4.59(brs), 4.67(s), 6.5(m), 6.78(m) and7.33(m).

Isomer (C/D) (2:1 ratio of diastereoisomers), m.p. 72°-76° C. (fromether). (Found: C,60.33; H,7.97; N,6.40. C₂₂ H₃₄ N₂ O₅ S requiresC,60.25; H,7.81; N,6.39%).

δ (CDCl₃): 0.95(m), 1.15(t,J=6 Hz), 1.3(m), 1.41(m), 1.75(m),2.01(0.66H,d,J=8 Hz), 2.21(0.33H,d,J=8H), 2.54(m), 2.66(m), 2.82(m),3.37(m), 3.72(s), 4.2(m), 4.38(m)), 4.6(m), 6.55(br.d,J=8 Hz) 6.7(m),6.9(br.s) and 7.35(m).

EXAMPLE 263-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)-carbonyl]-2-(3-indolyl)ethyl]amino]carbonyl]heptanoicacid, methyl ester (E26) ##STR58##

A solution of6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(3-indolyl)ethyl]amino]carbonyl]hept-2(and3)-enoic acid, methyl ester (D16; 2.5 g) in thiolacetic acid (100 ml)was left at room temperature for 28 days, then was evaporated to drynessin vacuo. Column chromatography (150 g) of the residue eluting initiallywith ether, and then ether-chloroform (1:1) gave the title compound(0.32 g) as a single isomer (Isomer A), m p. 134°-136° C. (from ether).

(Found C,60.32; H,6.88; N,8.80. C₂₄ H₃₃ N₃ O₅ S requires C,60.61;H,6.99; N,8.83%).

δ (CDCl₃): 0.84(d,J=5 Hz) and 0.85(d,J=5 Hz) (total 6H), 1.2-1.75(2H,m), 2.29(3H,s), 2.5-2.7(3H,m), 2.64(3H,d,J=5 Hz), 3.11(1H,dd,J=8,14Hz), 3.3(1H,dd,J=6,14 Hz), 3.62(3H,s), 3.94(1H,m), 4.72(1H,q,J=7 Hz),5.64(1H,brd), 6.52(1H,d,J=8 Hz), 7.05-7.25(3H,m), 7.36(1H,d,J=7 Hz),7.72(1H,d,J=7 Hz) and 8.17(1H,s).

EXAMPLE 273-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]2-(3-indolyl)ethyl]amino]carbonyl]heptanoicacid, methyl ester (E27) ##STR59##

An ice-cooled solution of3-acetylmercapto=6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(3-indolyl)ethyl]amino]carbonyl]heptanoicacid, methyl ester (E26; 0.1 g) in nitrogen-purged methanol (5 ml) wastreated with 35% aqueous ammonia (1.5 ml). The mixture was stirred atroom temperature under nitrogen for 2 h. The solution was evaporated todryness in vacuo and the residue was triturated with cold ether to givethe title compound as a single isomer (Isomer A), (52 mg), m.p. 73°-75°C.

(Found: C,60.69; H,7.35; N,9.64%. C₂₂ H₃₁ N₃ O₄ S requires C,60.95;H,7.21; N,9.69%).

δ (CDCl₃): 0.87(6H,d,J=6 Hz), 1.4-1.7(2H,m), 1.67(1H,d,J=9 Hz),2.37(1H,dd,J=5,15 Hz), 2.41(1H,m), 2.60(1H,dd,J=3,15 Hz), 2.65(3H,d,J=5Hz), 3.13(1H,dd,J=7,14 Hz), 3.21(1H,m), 3.30(1H,dd,J=7,14 Hz),3.67(3H,s), 4.75(1H,q,J=7 Hz), 5.68(1H,brd), 6.53(1H,d,J=8 Hz),7.05-7.25(3H,m), 7.35(1H,d,J=8 Hz), 7.71(1H,d,J=8 Hz) and 8.13(1H,s).

EXAMPLE 283-Acetylmercapto-N.-[1-(S)-[(methylamino)carbonyl]-2(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E28) ##STR60##

The title compound, m.p. 235°-240° C., was prepared from the compound ofExample 5 and ammonia by an analogous procedure to that described inExample 11.

EXAMPLE 293-Mercapto-N'-[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]-2-(2-methylpropyl)pentanediamide(E29) ##STR61##

The title compound, m.p. 221°-225° C., was prepared from the compound ofExample 28 by an analogous procedure to that described in Example 10.

EXAMPLE 303-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylglycine,ethyl ester (E30) ##STR62##

The title compound, m.p. 190°-198° C., was prepared from the compound ofExample 5 and ethylglycine hydrochloride by an analogous procedure tothat described in Example 12.

EXAMPLE 313-Mercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoylglycine,ethyl ester (E31) ##STR63##

The title compound, m.p. 221°-228° C., was prepared from the compound ofExample 30 by an analogous procedure to that described in Example 10.

EXAMPLE 323-Acetylmercapto-6-methyl-4-[[[1-(S)-(aminocarbonyl)2-phenylethyl]amino]carbonyl]heptanoicacid, methyl ester (E32) ##STR64##

The title compound was prepared from6-methyl-4-[[[1-(aminocarbonyl)-2-phenylethyl]amino)carbonyl]hept-2(and3)-enoic acid, methyl ester and thiolacetic acid by an analogousprocedure to that described in Example 26. Silica gel chromatographygave mixtures of diastereoisomers (Isomer A/B), m.p. 160°-164° C. and(Isomer C/D), m.p. 50°-54° C. (after trituration with ether). Theintermediate hept-2(and 3)-enoic acids were prepared from4-methoxycarbonyl-2-(2-methylpropyl)but-2-enoic acid and L-phenylalanineas described in Description 12.

EXAMPLE 333-Mercapto-6-methyl-4-[[[1-(S)-(aminocarbonyl)2-phenylethyl]amino]carbonyl]heptanoicacid, methyl ester (E33) ##STR65##

The title compound, isolated as a mixture of diastereoisomers, (IsomerA/B), and (Isomer C/D), m.p. 64°-66° C. was prepared from the compoundof Example 32 by an analogous procedure to that described in Example 10.The crude product was purified by silica gel chromatography using ethylacetate as the eluent.

EXAMPLE 343-Acetylmercapto-6-methyl-4-[[[1-(S)-[(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl-D-leucine,t-butyl ester (E34) ##STR66##

The title compound was prepared from the compound of Example 5 andt-butylleucine hydrochloride by an analogous procedure to that describedin Example 12 and exhibited a m.p. of 148°-157° C.

EXAMPLE 353-Acetylmercapto-6-methyl-4-[[[1-(S)-[(Methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl-D-leucine,(E35) ##STR67##

The title compound was prepared from the compound of Example 34 using ananalogous procedure to that described in Example 14 and exhibited a m.p.of 195°-200° C.

EXAMPLE 363-Mercapto-6-methyl-4-[[[1-(S)-(methylamino)carbonyl]-2-(4-methoxyphenyl)ethyl]amino]carbonyl]heptanoyl-D-leucine(E36) ##STR68##

The title compound was prepared from the compound of Example 35 using ananalogous procedure to that described in Example 15 and exhibited a m.p.of 216°-220° C.

EXAMPLE 37N-[N-[3Acetylmercapto-2-(2-methylpropyl)-1-oxo-4-[(phenylmethoxy)carbonyl]butyl]-L-leucyl-L-alanine,methyl ester. (E37) ##STR69##

The title compound was prepared from the compound of Description 7 by ananalogous procedure to that described in Example 32 usingL-leucyl-L-alanine, methyl ester. The title compound was isolated as ahygroscopic foam.

EXAMPLE 38N-[N-[3-Mercapto-2-(2-methylpropyl)-1-oxo-4-[(phenylmethoxy)carbonyl]butyl]-L-leucyl-L-alanine,methyl ester. (E38) ##STR70##

The title compound was prepared from the compound of Example 37 by ananalogous procedure to that described in Example 10. The title compoundwas isolated as a mixture of diastereoisomers, (Isomer A/B, 1:1), m.p.106°-110° C.

COLLAGENASE INHIBITOR ASSAY

The test is performed essentially as in Cawston and Barrett Anal.Biochem. 99, 340-345 (1979). Compounds for testing are dissolved inmethanol and added to purified rabbit bone collagenase in buffer. Aftera 5-15 min incubation at 37° C., the assay tubes are cooled to 4° C. and¹⁴ C-acetylated rat skin Type I collagen is added. The assay tubes areincubated at 37° C. overnight. The ¹⁴ C-collagen forms insoluble fibrilswhich are the substrate for the enzyme.

To terminate the assay, the assay tubes are spun at 12000 rpm for 15min. Undigested ¹⁴ C-collagen is pelleted, digested ¹⁴ C-collagen isfound as soluble peptides in the supernatant. A sample of thesupernatant is taken for liquid scintillation counting. The activity ofcollagenase inhibitors is expressed as that amount of compound whichinhibits a known amount of enzyme by 50% (IC₅₀) The activities ofrepresentative compounds of the invention, in the above test procedure,are illustrated in the table below:

    ______________________________________                                        Example No.                                                                              Isomer         IC.sub.50 (nM)                                      ______________________________________                                        2          mixture of     100                                                            diastereoisomers                                                   2          A              3.9-6.4 (2 expts).                                  2          B              72                                                  4          A              31                                                  4          B              270                                                 4          C              230                                                 4          D              12600                                               8          A              4.7-14 (3 expts.)                                   10         A              8.3                                                 ______________________________________                                    

We claim:
 1. A compound of the formula (I) or a salt, solvate or hydratethereof: ##STR71## in which: R₁ is --OH; alkoxy; aryloxy; aralkyloxy;amino, alkylamino, acyclic dialkylamino; or a group ##STR72## where R₈is hydrogen; alkyl optionally substituted by --OH, alkoxy, amino,alkylamino, acyclic dialkylamino, quanidine, --CO₂ H, CONH₂, SH orS-alkyl; or --CH₂ -Ar where Ar is optionally substituted aryl; and R₉ isalkoxy; OH; amino; alkylamino; or acyclic dialkylamino;R₂ is hydrogen;##STR73## where Z is optionally substituted aryl; R₃ is C₃₋₆ alkyl; R₄is hydrogen; alkyl; --CH₂ -R₁₀ where R₁₀ is optionally substitutedphenyl or 5- or 6- membered monocyclic or 9- or 10-membered bicyclicheteroaryl containing one or two heteroatoms selected from nitrogen,oxygen, and sulphur which in the case of there being more than oneheteroatom may be the same or different; or a group ##STR74## where R₁₁is hydrogen; alkyl; or --CH₂ -Ph where Ph is optionally substitutedphenyl; and R₁₂ is hydrogen or alkyl; and R₅ is hydrogen or alkyl; or agroup ##STR75## where R₁₃ is hydrogen; or alkyl; and R₁₄ is hydroxy;alkoxy; or --NR₆ R₇ where each of R₆ and R₇ is hydrogen or alkyl; or R₆and R₇ together with the nitrogen atom to which they are bonded form anN-morpholinyl or optionally nitrogen-substituted piperazinyl ring.
 2. Acompound according to claim 1 in which R₁ is selected from the groupconsisting of hydroxy, C₁₋₄ alkoxy, benzyloxy, amino, methylamino, and--NH--CH(R₈)COR₉ where R₈ is hydrogen or iso-butyl and R₉ is OH,NH₂,ethoxy or t-butoxy;R₂ is selected from the group consisting of hydrogen,acetyl, and benzoyl; R₃ is selected from the group consisting ofn-butyl, iso-butyl and sec butyl; R₄ is selected from the groupconsisting of iso-butyl, benzyl, 4-methoxy benzyl, 1-(benzyloxy)ethyland 3-indolylmethyl; and R₅ is selected from the group consisting ofhydrogen, methyl, and 1-(methoxycarbonyl)ethyl.
 3. A compound accordingto claim 1 in which R₁ is selected from methoxy, amino, methylamino or--NHCH₂ CO₂ H; R₂ is hydrogen, acetyl or benzyl 3-indolylmethyl; R₃ isiso-butyl; R₄ is benzyl or 4-methoxybenzyl and R₅ is methyl.
 4. Acompound according to claim 1 in which the chiral centre marked with anasterisk in formula (1) has the S configuration.
 5. A compound accordingto claim 1 which is selected from the group consistingof:3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoicacid, methyl ester, 3-Mercapto-6-methyl-4-(((I-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoic acid,methyl ester,3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoicacid,3-Mercapto-N5-methyl-N,(1-(S)-(methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)-pentanediamide,2-(3-Mercapto-6-methyl-4(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoylamino)ethanoic acid, N⁵-(Aminocarbonyl)methyl-3-mercapto-N'-(1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)pentanediamide,3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoic acid, benzyl ester,3-Acetylmercapto-6-methyl-4-(((-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoicacid, 3-Benzoylmercapto-N⁵ -methyl-N¹-(1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)-pentanediamide,3-Acetylmercapto-6-methyl-4-(((1(S)-((methylamino)-carbonyl)-2-phenylethyl)amino)carbonyl)heptanoic acid, methyl ester,3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-Carbonyl)-2-phenylethyl)amino)carbonyl)heptanoicacid, methyl ester, 3-Acetylmercapto-N⁵ -methyl-N¹-(1-(S)-((methylamino)carbonyl)-2-2(4-methyphenyl)ethyl)-2-(2-methyl-propyl)pentanediamide,2-(3-Acetylmercapto-6-methyl-4-((((S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl(heptanoylamino)ethannoicacid, t-butyl ester,2-(3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoylamino)ethanoic acid, t-butyl ester,2-(3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-amino)carbonyl)heptanoylamino)ethanoicacid, 3-Acetylmercapto-N⁵ -(aminocarbonyl)methyl-N¹-(1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)pentanediamide,1-(3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoyl)-4-methylpiperazine,1-(3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoyl)-4-methylpiperazine,1-(3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoyl)morpholinehydrate,1-(3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoyl)morpholine hemihydrate,3-Acetylmercapto-6-methyl-4-(((1-(SH)((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)-heptanoicacid, tert-butyl ester,3-Mercapto-6-methyl-4-(((-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)amino)carbonyl)heptanoicacid, tert-butyl ester,1-(3-Acetylmercapto-6-methyl-4-(((2-(R)-benzyloxy-1-(S)-((methylamino)carbonyl)propyl)amino)carbonyl)-heptanoicacid, methyl ester,3-Mercapto-6-methyl-4-(((2-(R)-benzyloxy-1-(S)-((methylamino)carbonyl)propyl)amino)carbonyl)heptanoicmethyl ester,3-Acetylmercapto-6-methyl-4-(((I-(S)-((methylamino)-carbonyl)-2-(3-indoyl)ethyl)amino)carbonyl)heptanoicacid, methyl ester,3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)carbonyl)-2-(3-indolyl)ethyl)amino)carbonyl)heptanoicacid, methyl ester,3-Acetylmercapto-N'-(1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)-pentanediamide,3-Mercapto-N'-(1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-2-(2-methylpropyl)-pentanediamide,3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)-carbonyl)heptanoylglycine,ethyl ester,3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)amino)-carbonyl)heptanoylglycine,ethyl ester,3-Acetylmercapto-6-methyl-4-(((1-(S)-(aminocarbonyl)-2-phenylethyl)amino)carbonyl)heptanoicacid, methyl ester,3-Mercapto-6-methyl-4-(((1-(S)-(aminocarbonyl)-2--phenylethyl)amino)carbonyl)heptanoicacid, methyl ester,3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-amino)carbonyl)heptanoyl-D-leucine,t-butyl ester,3-Acetylmercapto-6-methyl-4-(((1-(S)-((methylamino)carbonyl)-2-(4-methoxyphenyl)ethyl)-amino)carbonyl)heptanoyl-D-leucine,3-Mercapto-6-methyl-4-(((1-(S)-((methylamino)-carbonyl)-2-(4-methoxyphenyl)ethyl)-amino)carbonyl)heptanoyl-D-leucine,N-(N-(3-Acetylmercapto-2-(2-methylpropyl)-I-oxo-4-((phenylmethoxy)carbonyl)butyl)-L-leucyl-L-alaninemethyl ester.N-(N-(3-Mercapto-2-(2-methylpropyl)-1-oxo-4-((phenylmethoxy)carbonyl)butyl)-L-leucyl-L-alaninemethyl ester.
 6. A pharmaceutical composition for use in treatingcollagenolytic conditions comprising an effective amount of a compoundaccording to claim 1 and a pharmaceutically acceptable carrier.
 7. Amethod of treating collagenolytic conditions in mammals which comprisesadministering an effective amount of a compound according to claim 1 ora pharmaceutically acceptable salt, solvate or hydrate thereof, to asufferer.